Spore-forming bacterial species have a wide range of industrial applications including uses in agriculture, environmental remediation, composting, methane production, oil recovery, and cleaning supplies. Depending upon the particular bacterial species employed, compositions comprising the bacteria may be suitable for agricultural, horticultural, environmental, probiotic, aquatic, industrial and sanitation uses, among others. Several spore-forming bacteria species have utility across many industries. For example, Bacillus megaterium, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus subtilis, and Bacillus pumilus are used as agricultural fungicides, and in wastewater treatment, cleaning products, and direct fed microbials.
Regarding agricultural applications, the use of spore forming bacteria to enhance the health of plants is well known in the art. For example, Zhang et al. (2009, Plant J 58: 568-577) teach that the plant growth promoting bacterium Bacillus subtilis GB03 increases iron acquisition by plants and photosynthetic capacity. Xie et al. (2009, Plant Signaling and Behavior 4(10): 948-953) teach that volatiles produced by this same strain of Bacillus subtilis increased plant growth and seed count. Han et al. (2014, Frontiers in Plant Science 5(525): 1-8) report that white clover plants grown in Bacillus subtilis GB03 inoculated soil were significantly larger than non-inoculated controls with respect to shoot height, root length, plant biomass, leaf area and chlorophyll content. Spores of such bacteria may also be coated onto seeds or other plant propagative material, such that once sown or planted an enhanced environment which supports germination of the seed, stimulation of plant growth or biological protection of the seed and resulting plant can be established. For example, symbiotic bacteria such as those from the genera Rhizobium and Bradyrhizobium, which enable nitrogen fixation in leguminous plants may be used to inoculate leguminous plants to aid nodule formation. Inoculation can be accomplished by coating seeds, dusting on-farm of seeds or crops or placing inoculate in-furrow at planting time.
Spore forming bacteria may also be used in environmental remediation methods such as bioremediation. Bioremediation involves the use of microorganisms to convert chemical compounds into innocuous or less harmful chemical compounds. Bioremediation technologies generally have lower costs than competing physical technologies, and can be adapted to a broader range of contamination problems and variations in field conditions. See US 2002/0090697.
Contaminated media may be treated with bacteria to promote the biodegradation of organic contaminants in water, soil, and industrial wastes. For example, Lupton et al. (U.S. Pat. No. 5,062,956) discusses a method of removing soluble Cr(VI) using anaerobic bacteria to reduce Cr(VI) to Cr(III) and immobilize the Cr(III) as the insoluble hydroxide which settles out as a solid. Lupton et al. also describes treatment of aqueous residues containing undesirable amounts of Cr(VI) in a continuous bioreactor. Turick et al. (U.S. Pat. No. 5,681,739) describes a method of reducing the concentration of Cr(VI) in a liquid aqueous residue comprising the steps of providing Cr(Vi)-reducing bacteria, mixing the liquid aqueous residue with a nutrient medium (e.g., molasses, acetic acid, amino acids, casamino acids, urea), and contacting the mixture with the anaerobic bacteria to enhance the reduction of Cr(VI) to Cr(III). This process can be used for the bioremediation of hexavalent chromium contaminated soil and/or ground water.
One difficulty associated with the use of such bacteria is that while they must be in their vegetative state to afford such benefits, it is difficult to formulate Bacillus species and other spore forming bacteria in their vegetative form such that they will possess an adequate shelf life. In addition Bacillus species in their vegetative form may not be able to survive the harsh conditions needed for industrial uses, such as the conditions to which seeds or other plant propagative materials are subjected before conditions suitable for seed germination occur. In contrast, formulations of such species in their spore form are much more suitable for commercial and practical use. For example, as is noted by Cartman et al. (2008, Applied and Environmental Microbiology, August, p. 5254-5258) “[b]acterial spores are particularly well suited for use as live microbial products as they are metabolically dormant and highly resilient to environmental stress. These intrinsic properties are highly desirable from a commercial perspective and mean that spore-based products have a long shelf life and retain their viability during distribution and storage.”
The use of certain compounds, particularly certain L-amino acids, to stimulate the germination of Bacillus spores has been reported in the literature. For example, Foerster et al. (1996, Journal of Bacteriology 91(3): 1168-1177) discloses that the addition of L-alanine to spore suspensions in aqueous solutions will cause the germination of a number of Bacillus species. However one of the challenges in using combinations of bacterial spores and germinative compounds in industrial uses is maintaining the spore in an inactive form in the presence of the germinative compound until germination of the spores is required. For example, a plant propagative material such as a seed treated with a bacterial spore composition may be subjected to a storage period of several months before planting. Thus a need exists for bacterial spore formulations that can stimulate rapid germination of bacterial spores under favorable conditions, but that also prevent premature germination of the spores until germination of the spore is required.
In addition, bacteria for agricultural or industrial use may be exposed to a range of environmental conditions such as low soil pH, high salt concentrations (e.g. NaCl), and high metal concentrations (e.g. copper and aluminum) that are unfavorable for bacterial spore germination. Thus a need exists to develop formulations of bacterial spores that are able to germinate under these adverse conditions.